Skin encounters major challenges in maintaining genomic fidelity as its cells divide in the presence of environmental DNA damaging agents such as UVB and chemicals. When DNA is damaged or DNA synthesis is otherwise delayed, a cell requires additional time to ensure complete DNA replication. The replication checkpoint is the mechanism by which a cell ensures that DNA replication is complete prior to initiating chromatin condensation, an initial stage of mitosis. We have shown that the protein kinase ATR and its downstream target, Chk-1, are required for the replication checkpoint and for survival after DNA damage. Critical to this proposal is that pre-malignant and cancer cells are deficient in p53 and other early cell cycle checkpoints and are thus highly sensitive to inhibition of ATR, leading to cell death. Complete loss of function of the replication checkpoint by genetic deletion of either ATR or Chk-1 is rapidly lethal to a developing embryo or to dividing cells in culture. In contrast, partial inhibition of the replication checkpoint by small molecule inhibitors or by expression of dominant negative ATR or Chk-1 is tolerated for many cell divisions in unstressed cells. The precise consequences of partially inhibiting the replication checkpoint are not known for normal or DNA damaged cells in vivo. The broad, long-term objectives of this proposal are: i) To further elucidate the molecular mechanism of the replication checkpoint in maintaining genomic fidelity, ii) To define the in vivo role of the replication checkpoint using skin as a model organ system, iii) To determine whether inhibiting the replication checkpoint can be used to eliminate DNA-damaged cells at risk of malignant progression. We will achieve these objectives with a variety of approaches including in vitro studies of chromosomal fragile sites, transgenic mice with diminished replication checkpoint function in skin, and topical application of small molecule inhibitors of the replication checkpoint to UVB-damaged mouse skin. This proposal will define the molecular mechanisms of the replication checkpoint in maintaining genomic integrity in a model organ system (mouse skin) and investigate its inhibition as a selective means of eliminating DNA-damaged pre-malignant cells in vivo.